The multifunctional enzyme Flap EndoNuclease (FEN-1 or FEN) and the homologous 5' family nuclease XPG play key but incompletely understood roles in DNA replication, repair, and genome integrity. FEN and XPG activities are furthermore aided by their partner protein, the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen (PCNA). This proposal has three Aims: (1) characterize FEN and XPG substrate interactions, (2) elucidate FEN and XPG nuclease mechanisms and conformational changes, and (3) determine PCNA interactions and roles in FEN, XPG, and ligase activities including handoffs, which we propose avoid the release of toxic DNA intermediates in DNA replication and repair pathways. The Aims will test our hypotheses on mechanisms for DNA recognition, for structural rearrangements to position the DNA in the active site for catalysis and for PCNA-mediated coordination rather than interference between FEN and ligase bound to PCNA and between XPG and the p21 inhibitor of PCNA binding by polymerase. To accomplish the Aims, we will combine the complementary techniques of small angle X-ray scattering (SAXS) for solution structures and macromolecular X-ray crystallography (MX) for high resolution, with informed biochemical and mutational experiments. Targeted experiments on human and archaeal proteins will allow efficient and systematic analyses fundamental to understanding FEN, XPG, and ligase activities plus their functional PCNA interactions. Quantitative characterizations by SAXS and MX along with biophysical and mutational analyses in the Tainer lab will be aided by ongoing collaborative research on FEN biochemistry, FEN inhibition, and XPG activities plus single molecule fluorescence technologies applied to PCNA complexes. The expected results will clarify the molecular determinants for 5' family DNA interactions and nuclease activities including FEN, XPG, and ligase interactions with PCNA relevant to DNA replication, base repair, and nucleotide excision repair. Overall, this research will provide a fundamental molecular framework for FEN, XPG, ligase and PCNA activities and consequent roles in the regulation of genome fidelity. The results will also clarify mechanisms whereby loss of the functions of these coordinated complexes, which act in DNA replication and repair processes, may lead to inheritable genetic defects and the cancer initiation.